Interaction of AcMADS68 with transcription factors regulates anthocyanin biosynthesis in red-fleshed kiwifruit

Abstract In red-fleshed kiwifruit, anthocyanin pigmentation is a crucial commercial trait. The MYB-bHLH-WD40 (MBW) complex and other transcription factors regulate its accumulation. Herein, a new SEP gene, AcMADS68, was identified as a regulatory candidate for anthocyanin biosynthesis in the kiwifruit by transcriptome data and bioinformatic analyses. AcMADS68 alone could not induce the accumulation of anthocyanin both in Actinidia arguta fruit and tobacco leaves. However, in combination with AcMYBF110, AcMYB123, and AcbHLH1, AcMADS68 co-overexpression increased anthocyanin biosynthesis, whereas its silencing reduced anthocyanin accumulation. The results of the dual-luciferase reporter, firefly luciferase complementation, yeast two-hybrid and co-immunoprecipitation assays showed that AcMADS68 could interact with both AcMYBF110 and AcMYB123 but not with AcbHLH1, thereby co-regulating anthocyanin biosynthesis by promoting the activation of the target genes, including AcANS, AcF3GT1, and AcGST1. Moreover, AcMADS68 also could activate the promoter of AcbHLH1 surported by dual-luciferase reporter and yeast one-hybrid assays, thereby further amplifying the regulation signals from the MBW complex, thus resulting in enhanced anthocyanin accumulation in the kiwifruit. These findings may facilitate better elucidation of various regulatory mechanisms underlying anthocyanin accumulation and contribute to the quality enhancement of red-fleshed kiwifruit.


Introduction
Kiwifruit (Actinidia, Actinidiaceae) is a popular fruit worldwide, and the red-f leshed kiwifruit is famous amongst consumers for its brilliant color and comparatively anthocyanin abundance [1]. Anthocyanins occur ubiquitously in plant tissues, wherein these are crucial for seed dispersal, pollination, protection against pathogens, and regulating responses to environmental stress [2,3]. Moderate to high levels of anthocyanins is a crucial fruit trait for human nutrition owing to the significant antioxidant effects and potential health benefits of these molecules [4][5][6].
In kiwifruit, structural genes of anthocyanin biosynthesis have been well studied [27][28][29][30][31]. Several R2R3MYB TFs, including AcMYB5-1, AcMYBA1-1, AcMYB1, and AcMYB75, are correlated with anthocyanin accumulation in the fruit [32,33]. Wang et al. demonstrate that AcbHLH42 and AcMYB123, which are homologs of AtTT8 and AtTT2, respectively, participate in regulating anthocyanin biosynthesis by altering the expression levels of AcF3GT1 as well as AcANS [34]. AcMYBF110, a homolog of AcMYB110 that is specifically implicated in the red petals of kiwifruit, exhibits characteristic expression in fruit and can autonomously induce accumulation of anthocyanin in tobacco leaves by up-regulating the expressions of DFR, ANS, and UFGT [1,35]. Recently, four new regulators -AcbHLH1, AcbHLH5, AcbHLH4 and AcWDR1 -have been identified in kiwifruit. These four regulators interact with AcMYBF110, resulting in the formation of three MBW complexes, thereby directly or indirectly regulating anthocyanin biosynthesis [36]. Wang et al. suggest that differential expression and subsequent repression of MYB110 and MYB10 by SPL/ARF/SCL are responsible for differential anthocyanin accumulation in kiwifruit species [37]. However, other upstream regulatory genes involved in the accumulation of anthocyanin in the kiwifruit remain largely unknown.
In this study, transcriptome data from four inner pericarp stages and expression profiles during different developmental stages were analysed. The expression of the SEP MADS gene, AcMADS68 (GenBank accession number ON175934) correlated remarkably with anthocyanin accumulation in the kiwifruit. Its function was confirmed by the transient transformation in Actinidia arguta fruit and tobacco leaves. Firef ly luciferase complementation, yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) assays validated the interaction of AcMADS68 with AcMYBF110 or AcMYB123. Finally, the activation of anthocyaninrelated gene transcription by AcMADS68 and its partners was evaluated by a dual-luciferase reporter assay and Y1H. Our findings reveal a potential regulatory mechanism via which AcMADS68 inf luences kiwifruit anthocyanin biosynthesis, and may have implications for the regulatory network that underlies anthocyanin biosynthesis in other plants.

Color and anthocyanin content in developing red-fleshed kiwifruit
The f lesh color of both outer and inner pericarp was green at the first fruit developmental stage [S1, 65 days after pollination (DAP), Fig. 1a] of Actinidia chinensis 'Hongyang'. The f lesh color of the inner pericarp changed to red in S2 (90 DAP), whereas that of the outer pericarp remained green until maturity to harvest (S4, 140 DAP). Hue angle (90 < h • < 180, green or yellow; 0 < h • < 90, red) decreased dramatically in the inner pericarp during development but the outer pericarp showed lesser changes (Fig. 1b). Cyanidin 3-O-xylogalactoside (Cy-xyl-gal) [1] is a major anthocyanin molecule, accounting for 82.36% of the total anthocyanins (Fig. 1c). As expected, no or only trace levels of anthocyanins were detected in S1, both in the outer and inner pericarp (Fig. 1b). Anthocyanins began to accumulate rapidly from S2 until S4 in the inner pericarp but the levels in the outer pericarp altered negligibly and were substantially lower relative to the former.

Transcriptome analysis for fruit developmental stages
The regulatory network for anthocyanin accumulation during kiwifruit development, that is, for the four inner pericarp stages (S1 to S4) was obtained from an RNA-seq study. The numbers of clean reads (Q30 88.58%) in all the 12 samples ranged from 19 262 389 to 28 959 486, and 91.02%-96.00% of these reads could be mapped to the kiwifruit reference genome [38,39] (Table S1, see online supplementary material). Transcriptome characteristics of biological replicates from the same stage (Fig. S1, Table S2, see online supplementary material) were highly correlated (r 2 = 0.908-0.999), indicating the reproducibility of transcriptome data and that these could be used for further analysis. Results from the principal component analysis showed that the first two principal components could explain 59.7% and 33.4% of the variance among the samples, respectively (Fig. 1d). Green (S1) and red samples (S2-S4) were separated by PC2.

Comparison of differentially expressed genes (DEGs) across developmental stages
Comparisons were made between S1 and S2 to S4 to identify the DEGs upon anthocyanin accumulation in the kiwifruit (Fig. 1e). A total of 3843, 4640, and 4361 DEGs were identified in the three corresponding comparisons, 'S1 vs. S2', 'S1 vs. S3', and 'S1 vs. S4'. A total of 2464 DEGs were commonly shared among the three comparisons, indicating a continued function of DEGs during all developmental stages. Upregulated DEGs were significantly higher relative to the down-regulated DEGs across comparisons (Figs 1e and S2, see online supplementary material). KEGG analysis indicated significant enrichment of the DEGs in 'plant hormone signal transduction', 'phenylalanine metabolism', and 'f lavonoid biosynthesis' (Fig. 1f-h). These biological processes are closely associated with f lavonoid biosynthesis [40]. Our results provide insight into the biological functions of the identified DEGs.

Identification of putative TFs involved in the accumulation of anthocyanins
A trend analysis was performed using 1407 DEGs (FPKM >0.5, listed in Table S3, see online supplementary material) obtained from the three comparisons to identify the putative TFs that could regulate anthocyanin accumulation during kiwifruit development. Interestingly, profile 19 (103 genes) showed changes in expression that were most similar to those observed in anthocyanin accumulation of the developing inner pericarp (Figs 1b and 2a; Fig. S3, see online supplementary material). Twelve of the genes in profile 19 were identified as TFs, and these included MYB, NAC, and MADS TFs (Table S4 and Fig. S4, see online supplementary material). Among them, AcMYBF110 (kiwifruit_newgene_50) plays a crucial role in the accumulation of anthocyanins in kiwifruit [1,36]. Results of qPCR indicated that Achn196271 expression correlated remarkably with those of anthocyanin-related genes, AcMYBF110, AcF3GT1 [30], and AcGST1 [29], in the inner and outer pericarp during fruit development; the highest correlation was observed with anthocyanin accumulation (r = 0.83, P < 0.01, Fig. 2b and c; Table S5, see online supplementary material). In contrast, lower correlations were observed for other candidate TFs (|r| = 0.01 to 0.58). These findings suggested that Achn196271 may serve as an important TF involved in the accumulation of anthocyanins in kiwifruit. S2' (f), 'S1 vs. S3' (g), and 'S1 vs. S4' (h). The genes that were up-regulated are shown by the red arrows, while those that were down-regulated are denoted by the green arrows in the (e). IP, inner pericarp; OP, outer pericarp. Stages S1-S4 represent 65, 90, 115, and 140 DAP.

Isolation and expression of AcMADS68
In total, 69 MADS TFs were obtained in the A. chinensis Hongyang and Red5 genome database [38,39]. Achn196271 (Acc32725) was named AcMADS68 based on its location on the chromosome (Table S6, see online supplementary material). The full-length AcMADS68 sequence was successfully isolated and cloned from the 'Hongyang' fruit. This encoded a protein of 245 amino acids (Fig. 3a). Phylogenetic analysis showed that AcMADS68 was grouped in the same clade as MdMADS18 and PyMADS18 (Fig. S5, see online supplementary material), two potential regulators involved in anthocyanin biosynthesis [25,41]. Sequence alignment confirmed the presence of the four typical plant-MADS regions -the C-terminal domain, K box, I region, and M domainin AcMADS68 and other plant anthocyanin-related MADS TFs (Fig. 3a) [42]. AcMADS68 shared a higher amino acid sequence identity with MdMADS18 (81.63%) and PyMADS18 (80.08%) but its identity was lower with IbMADS10 and VmTDR4 (44.44% and 40.08, respectively).
The transcript levels were measured in the fruits of several red-f leshed kiwifruit cultivars and across tissue types of 'Hongyang', to obtain the spatial and temporal expression profiles of AcMADS68. In the five red-f leshed kiwifruits, transcript abundance of AcMADS68 was elevated in the inner pericarp relative to the outer pericarp, consistent with the distribution of anthocyanins (Fig. 3b); while in two green cultivars, lower transcript levels were found in bothe outer and inner pericarp. AcMADS68 transcript levels and anthocyanin content were remarkably higher in the ovary relative to petals, stems, and leaves, wherein anthocyanin was negligibly present (Fig. 3c). Given the above data, AcMADS68 expression corre-lated substantially with kiwifruit coloration and anthocyanin accumulation.

Subcellular localization and transcriptional activity of AcMADS68
The 35S:AcMADS68-GFP fusion protein was transiently expressed in six-week-old Nicotiana benthamiana leaves to determine the subcellular localization of AcMADS68. The 35S:GFP protein localized to both the cytoplasm and nucleus, whereas the 35S:AcMADS68-GFP protein was exclusively detected in the nucleus, suggesting that AcMADS68 was a nuclear protein as shown in Fig. 3d.
Reporter vectors comprising 5× GAL4 activation domains upstream of the LUC gene and the effector vector (pBD, pBD-AcMADS68, and pBD-VP16) were transiently co-expressed in N. benthamiana leaves [43] to assess AcMADS68's transcriptional activity in vivo. As with the positive control, pBD-VP16, the LUC/REN ratio increased significantly upon reporter vector's coexpression with pBD-AcMADS68 as compared to the negative control pBD (Fig. 3e). These data suggested that AcMADS68 may function as a transcriptional activator.

Heterologous overexpression of AcMADS68 and related TFs induces anthocyanin biosynthesis in tobacco leaves
First, AcMADS68's function was tested by the transient transformation of tobacco (Fig. 4a). After seven days, no pigmentation was detected in leaves infiltrated with the empty vector, AcMADS68, AcMYB123, or AcMYB123 + AcbHLH1 (data not shown) alone. By contrast, obvious pigmentation was found at the infiltration sites transformed with AcMYBF110 + AcbHLH1, AcMYBF110, or AcMYB123 + AcbHLH1. Notably, stronger pigmentation was seen when AcMADS68 was co-infiltrated with the above three constructs. The anthocyanin content and transcript abundances of anthocyanin-related genes, including NtDFR, NtANS, and NtUFGT, were consistent with the visual phenotypes (Fig. 4b).

Overexpression of AcMADS68 and related TFs cause anthocyanin biosynthesis in kiwifruit
To further confirm the function of AcMADS68 in anthocyanin biosynthesis in kiwifruit, the immature A. arguta fruit was subjected to transient transformation with AcMADS68 and anthocyanin-related TFs [36]. No obvious pigmentation was observed after injection with the following negative controls: 35S, 35S:MADS68, TRV1 + TRV2, and TRV1 + TRV2-MADS68 (Fig. 5a). Significantly higher levels of anthocyanin accumulated in fruits co-expressing AcMYBF110 or AcMYB123 with AcbHLH1 (Fig. 5a-c). Much stronger pigmentation was observed when the two combinations, AcMYBF110 + AcbHLH1 and AcMYB123 + AcbHLH1, were co-expressed with AcMADS68. Reduced pigmentation was observed when these were co-expressed with the RNAi vector, TRV1 + TRV2-MADS68. The transcript levels of anthocyaninrelated genes showed similar trends. Higher levels were observed when AcMYBF110 + AcbHLH1 or AcMYB123 + AcbHLH1 was coexpressed with AcMADS68; these were lower when AcMYBF110 + AcbHLH1 or AcMYB123 + AcbHLH1 was co-expressed with TRV1 + TRV2-MADS68 ( Fig. 5e-g). Moreover, the expression of AcbHLH1 was up-regulated upon AcMADS68 overexpression (Fig. 5d), while those of AcMYBF110 and AcMYB123 remained unchanged (Fig. S6, see online supplementary material). These findings indicated that AcMADS68 participated in the regulation of anthocyanin biosynthesis in kiwifruit.

Interaction of AcMADS68 with other anthocyanin-related TFs
Full-length AcMADS68 cDNA or AcMADS68 with different Nor C-terminal deletions was cloned into pGBKT7 (Fig. 6a) to test its interactions with AcMYBF110, AcMYB123, or AcbHLH1, inserted into pGADT7. The complete amino acid sequence (VII, 1-245), N-terminal fragment IV (1-178, M domain + I region + K box), fragment V (116-178, K2-K3 box), and the C-terminal fragment (VI, 60-245) showed strong transcriptional activity in yeast (Fig. 6b). However, no transcriptional activity was found for the three N-terminal fragments, namely fragments I (the conserved M domain, residues 1-59), II (M domain + I region, 1-87), and III (M domain + I region + K1 box, 1-116). These results suggested that AcMADS68 contains a large activation domain in the K2-K3 region of its typical K-box [44,45]. To avoid false-positive results, MADS68 1-59 (I), MADS68 1-87 (II), and MADS68 1-116 (III) with no transcriptional activity were selected for Y2H analysis. When fragment I or II of AcMADS68 was co-transformed with AcMYBF110 or AcMYB123, growth was observed on SD/−T/−L medium but not on SD/−T/−L/−A/-H medium (Fig. 6c). Interestingly, when fragment III (MADS68 1-116 ) was co-transformed with AcMYBF110 or AcMYB123, the yeast grew on both the SD/−T/−L medium and the SD/−T/−L/−A/-HA medium. Thus, the site of AcMADS68 interaction with AcMYBF110 or AcMYB123 was within the amino acid sequence of the K1 region (87-116). When AcbHLH1 was co-transformed with fragments I, II, or III of AcMADS68, no yeast growth was observed on the SD/−T/−L/−A/-HA medium (Fig. 6c), indicating that AcMADS68 could not interact with AcbHLH1. Collectively, these findings suggested that, in yeast, AcMADS68 interacted with the two MYB TFs (AcMYBF110 and AcMYB123) but not with AcbHLH1.
Tobacco-based firef ly luciferase complementation assays were performed to confirm the results recorded from Y2H assays (Fig. 6d). Relative to the five negative controls, luciferase activity increased when NLuc-MADS68 was co-expressed with AcMYBF110-Cluc or AcMYB123-Cluc, and the combination of Nluc-MADS68 with AcMYBF110-Cluc showed the highest activity (Fig. 6d). Co-transformation of Nluc-MADS68 with AcbHLH1-Cluc resulted in lower luciferase levels, which did not differ significantly from those of the controls. These results were in line with the findings from the Y2H assays.
An in vivo co-immunoprecipitation (Co-IP) assay also confirmed that AcMYBF110 and AcMYB123 could be coimmunoprecipitated by AcMADS68 in total N. benthamiana leaf extracts (Fig. 6e). Figure 6. Interaction of AcMADS68 with other anthocyanin-related TFs. a I-VII represents distinct amino acid residues of AcMADS68. b Self-activation of AcMADS68 transcriptional activity. c Yeast-two-hybrid assay. AD, activation domain; BD, DNA-binding domain; SD/−TL, SD/Trp-Leu medium; SD/-TLAH, SD/−Trp-Leu-Ade-His medium. d Firef ly luciferase complementation assay in N. benthamiana leaves. Activation of anthocyanin-related gene promoters by AcMADS68. e Co-IP assays showing that AcMYBF110-FLAG and AcMYB123-FLAG, respectively, co-immunoprecipitate with AcMADS68-GFP in N. benthamiana leaf. IB, immunoblotting. f Different combinations of promoter activation of AcANS, AcF3GT1, and AcGST1. g Activation of AcMYBF110, AcMYB123, and AcbHLH1 promoters by AcMADS68. h AcMADS68 binding to the AcbHLH1 promoter was shown using a Y1H assay. The prey vector AD-AcMADS68 contained AcMADS68, whereas the AD-empty vector was employed as a negative control. SD/−Leu, SD medium without Leu; SD/−Leu/AbA 400 , SD medium without Leu augmented with 400 ng mL −1 AbA. i AcMADS68 stimulates the activity of five distinct AcbHLH1 promoter segments. Mean standard errors are represented by the error bars (n = 3). Variations that reach the significance threshold of P < 0.05 are denoted by the use of lowercase letters (one-way ANOVA). * P < 0.05, * * P < 0.01, * * * P < 0.001 (Student's t-test).

Activation of anthocyanin-related gene promoters by MADS68-MYBF110-bHLH1 and MADS68-MYB123-bHLH1 complexes
To assess the interaction of AcMADS68 with the promoters of AcANS, AcF3GT1, and AcGST1, a dual luciferase assay was performed in tobacco leaves (Fig. 6f). None promoter could be activated when AcMADS68 was expressed alone relative to the empty vector (SK). All three promoters were clearly activated upon AcMYBF110 expression alone and upon co-expression of AcMYBF110 + AcbHLH1 or AcMYB123 + AcbHLH1. Interestingly, the activation effects were enhanced by co-expression with AcMADS68. These findings indicated that the MADS68-MYBF110-bHLH1 or MADS68-MYB123-bHLH1 complex could directly activate the promoters of AcANS, AcF3GT1, and AcGST1, resulting in the accumulation of anthocyanins in the kiwifruit.
A dual-luciferase and Y1H assay demonstrated that the promoter of AcbHLH1I, but not AcMYBF110 or AcMYB123, was activated directly by AcMADS68 (Fig. 6g-h). To unravel the roles of different CArG motifs in the AcbHLH1 promoter, five different fragments (P0 to P4) were constructed to perform dual-luciferase assays (Fig. 6i). For the promoter fragment P4 that lacked the CArG motif, no obvious activation was observed. All of the other promoter fragments (P0-P3), comprising different numbers of CArG motifs showed activation to various extents.
In kiwifruit, several identified TFs are known to function as regulators of anthocyanin biosynthesis. AcMYBA1-1 and AcMYB5-1 enhance anthocyanin accumulation at low storage temperatures by upregulating the expression of genes in the anthocyanin pathway [32]. In Arabidopsis, overexpression of AcMYB75, whose expression during the fruit development of 'Hongyang', correlates remarkably with anthocyanin accumulation, can enhance anthocyanin accumulation [33]. AcMYB123, interacting with AcbHLH42, regulates anthocyanin accumulation by activating the promoter of AcF3GT1 in kiwifruit [34]. Herein, AcMYBF110 was found to play a crucial regulatory role in anthocyanin accumulation via the activation of the promoters of several genes in the anthocyanin pathway, especially AcANS, AcF3GT1, and AcGST1. AcMYBF110 interacts with both AcWDR1 and AcbHLH1/4/5, thereby forming three distinct MBW complexes that hierarchically regulate anthocyanin biosynthesis in the red-f leshed kiwifruit [36]. Overall, these reports focus on the functional regulation of MYB TFs in anthocyanin biosynthesis in the kiwifruit.
Transcriptomics has been widely used for identifying potential regulators of fruit quality traits in horticultural crops [59][60][61][62]. In this study, RNA-seq data, trend analysis, and expression profiles during multiple developmental stages helped us newly identify the SEP MADS TF, AcMADS68 (Figs 1 and 2). The expression of AcMADS68 correlated remarkably with color and the accumulation of anthocyanins in kiwifruit (Fig. 3b and c). AcMADS68's function was also confirmed by transiently transforming A. arguta fruit and tobacco leaves (Figs 4 and 5). Thus, we characterized the involvement of AcMADS68 in the regulation of anthocyanin biosynthesis in kiwifruit.

The interaction of AcMADS68 with AcMYBF110 and AcMYB123 regulates anthocyanin biosynthesis
A regulatory network incorporating TFs and the MBW complex controls anthocyanin biosynthesis [8,9]. Several TFs can regulate anthocyanin biosynthesis by promoting the activation of positive MYB or MBW complexes. For example, FaRAV1 enhances the accumulation of anthocyanin by the activation of genes in the anthocyanin pathway and FaMYB10 [16]. This finding is similar to those reported for peach BL [17], apple MdHY5 and MdHB1 [21,22], and pear BBX16 [20]. Some anthocyanin activators, including Arabidopsis AtTCP3 [63], apple MdERF1B and MdERF38 [50], and pear PyERF3 and PyWRKY26 [18,56], typically interact with anthocyanin-activating MYBs and enhance the activation of MBW complexes, thereby promoting anthocyanin accumulation. In addition, a few TFs, including PpMYB18, MdMYB16/17, and AtMYBL2 [64][65][66][67], regulate anthocyanin accumulation by competing with MYB or bHLH, thus interrupting the MBW complex. Herein, the results of firef ly luciferase complementation and Y2H assays demonstrated that AcMADS68 interacted separately with AcMYBF110 and AcMYB123 but not with AcbHLH1 (Fig. 6a-e). AcMADS68 did not activate the promoter of AcMYBF110 or AcMYB123 (Fig. 6h-i). This result differs from that reported previously by Jaakola et al., whereby VmTDR4 attenuated anthocyanin accumulation, either by indirect or direct control of the R2R3 MYB family during ripening of bilberry [24]. Phylogenetic analysis suggested that AcMADS68 was a SEP MADS gene, and VmTDR4 belonged to the AP1 subfamily (Fig. S5, see online supplementary material). They were placed in different clades and shared 40.08% identity (Fig. 3a), indicating differential regulation of anthocyanin, similar to other functions in plants [68]. This is supported by the differential regulation of anthocyanin accumulation by FaRAV1 and FaERF85 in strawberry [16].
The MBW complex and other anthocyanin-related TFs in several plants promote anthocyanin accumulation by stimulating gene expression in the anthocyanin pathway [9]. As shown in Figs 4 and 5, genes in the anthocyanin pathway were significantly upregulated when AcMADS68 was co-expressed with AcMYBF110, AcMYBF110 + AcbHLH1, or AcMYB123 + AcbHLH1 in A. arguta fruit or tobacco leaves, which was consistent with the results of anthocyanin accumulation. Dual-luciferase reporter assays showed that promoter activation of AcANS, AcF3GT1, and AcGST1 was strongly enhanced by overexpression of the AcMADS68-AcMYBF110-AcbHLH1 or AcMADS68-AcMYB123-AcbHLH1 complex (Fig. 6f). Hierarchical and feedback mechanisms between the MBW complex was an important feature of anthocyanin regulation [8,9,36]. For example, overexpression of AtPAP1/AtTT2 Figure 7. Anthocyanin biosynthesis of kiwifruit is regulated by AcMADS68 in two ways. Firstly, AcMADS68 interacted separately with AcMYBF110 and AcMYB123 resluting in stabilizing the formation of the MBW complex, thus promoting anthocyanin biosynthesis by upregulating the expression of anthocyanin related genes. On the other hand, the promoter of AcbHLH1 was directly activated by AcMADS68 and then amplified the MBW complex's regulation signals, resulting in enhanced accumulation of anthocyanins in kiwifruit.
In this study, the TF AcMADS68, which interacted separately with AcMYBF110 and AcMYB123, stabilized the formation of the MBW complex and upregulated the expression of AcANS, AcF3GT1, and AcGST1, thus promoting anthocyanin biosynthesis (Fig. 7). AcMADS68 directly activated the promoter of AcbHLH1, thus amplifying the MBW complex's regulation signals and resulting in enhanced accumulation of anthocyanins in the red-f leshed kiwifruit. These findings provide novel insight into the regulatory network of anthocyanin biosynthesis and may contribute to the production of new kiwifruit cultivars with high anthocyanin abundance.

RNA-seq and data analyses
RNA library preparation and sequencing were performed as described previously [69] using inner pericarps at the four developmental stages (S1 to S4). The concentration, integrity, and purity of the resulting RNA were detected with appropriate instruments and methods. HISAT2 v2.0.5 and HTSeq v0.7.2 were used to align and count the clean reads to the kiwifruit reference genome, and the FPKM values were estimated from the read counts mapped onto the genes and the gene lengths. DESeq2 package in R (1.18.0) was used to analyse identified significant DEGs with the threshold fixed at fold change >2 and P < 0.05 [70,71].
The omicshare website (www.omicshare.com/tools/Home/ Soft/pathwaygseasenior) was used to conduct a statistical analysis of the enrichment of DEGs in KEGG pathways. Among them, 1407 genes (FPKM>0.5) were subjected to trend analysis (www.omicshare.com/tools/Home/Soft/trend).

Expression analyses by qPCR
The Plant RNA Kit (Omega Biotek, Norcross, GA, USA) was employed to isolate around 1 μg of total RNA, which was then subjected to cDNA synthesis utilizing the Prime Script RT reagent kit (TaKaRa, Dalian, China). Additionally, qPCR was performed with the SYBR Premix Ex TaqII Kit (TaKaRa, Dalian, China). The conditions for amplification entailed one cycle at 95 • C for 40 s followed by 40 cycles at 95 • C for 30 s, and 58 • C for 30 s. Primers applied in this research are detailed in Table S7 (see online supplementary material). By employing the 2 − Ct method, data were normalized relative to actin and PP2A [72]. We performed each analysis three times (three PCRs for each biological duplicate, and three times for each gene in the sample) with biological replicates.

Extraction of AcMADS68 and sequence analyses
The whole set of 69 MADS TF sequences was derived from the kiwifruit genome database (http://kiwifruitgenome.org/home). After matching the protein sequences with ClustalW and making any required adjustments manually, a phylogenetic tree was generated in MEGA 5.0 utilizing the neighbor-joining technique and 1000 bootstrap iterations [73].

AcMADS68 subcellular localization
The coding region of AcMADS68 that did not include the stop codon was subjected to PCR amplification, after which it was inserted into the pCAMBIA2300 vector in frame with the GFP sequence (Table S7 contains a listing of the primer sequences, see online supplementary material). The fused constructs, 35S:AcMADS68-GFP and 35S:GFP, were transmuted into A. tumefaciens strain, GV3101. A. tumefaciens strain (OD 600 = 0.6-0.8) was individually introduced into leaves of N. benthamiana that are six weeks old. Using a confocal laser scanning microscope (TCS SP8 SR, Leica) the intensity of the f luorescence signal was evaluated 48 hours following a transformation.

Transient luciferase assays
The reporter, which is a derivation of pGreenII 0800-LUC and contains 5× GAL4 AD upstream of the minimum CaMV35S promoter as well as the LUC gene, was utilized for determining the level of transcriptional activity (Fig. 3e). This effector vector, pBD-AcMADS68, was generated by cloning the AcMADS68 coding sequence (CDS) into the pBD vector, which contains the GAL4 DNA-binding site. The negative control was the pBD (empty vector) whereas pBD-VP16 was a positive control.
The CDSs of AcMADS68, as well as those of other TFs, were introduced into the pGreenII 62-SK vector, resulting in the formation of the effectors [74] for DNA-promoter interaction assays (Table S7 outlines the primer sequences, see online supplementary material), and the reporters were obtained by inserting the promoters of genes associated with anthocyanin into the pGreenII 0800-LUC vector. As a negative control, we used the SK vector.
With the pSoup helper plasmid, each of the generated constructs was transferred into the GV3101 strain of A. tumefaciens. Injections of several Agrobacterium mixtures (OD600 0.8) containing the reporter and effectors were made into the leaves of N. benthamiana that had been growing for six weeks. Fortyeight hours following the transformation, luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA) in compliance with the specifications provided by the manufacturer. To conduct a dualluciferase experiment, the leaves that had been transformed were both sprayed and immersed in a solution consisting of 1.0 mM luciferin (Promega) in 0.01% Triton X-100. After five minutes of being in the dark, the f luorescence was quenched, and the LUC pictures were acquired utilizing a low-light CCD imaging device that was cooled (Lumazone Pylon2048B; Roper Scientific, Trenton, NJ, USA).

Transient expression in A. arguta fruit and tobacco leaves
By using the pSoup helper plasmid, the pGreenII 62SK-AcMADS68, AcMYBF110, AcMYB123, and AcbHLH1, and pGreenII 62-SK empty vector were introduced into the GV3101 A. tumefaciens strain for transient overexpression. The TRV2-MADS68 construct was created by inserting a coding sequence from AcMADS68 into pTRV2 to silence gene expression. Various combinations of these strains (about 400 μL) were introduced into either N. tabacum leaves or immature A. arguta fruit. The injected tobacco leaves were sampled seven days after transformation and photographed. The infiltrated A. arguta fruit was left on the vine for an additional ten days, imaged, and harvested for measuring anthocyanin content and extracting RNA.

Y2H assays
The full-length CDS of AcMADS68 and six partial fragments of AcMADS68 (MADS68 1-59 , MADS68 1-87 , MADS68 1-116 , MADS68 1-178 , MADS68 116-87 , and MADS68 60-245 ) were inserted into pDBKT7. The resulting constructs were transformed into Y2HGold with the Matchmaker Gold Yeast Two-Hybrid System (Clontech, Mountain View, CA, USA). The transformed yeast cells were cultured on SD/−Leu/−Trp/-His/−Ade and SD-Trp-Leu media to assess the transactivation of AcMADS68. The full-length CDSs of AcMYBF110, AcMYB123, and AcbHLH1 were inserted into pGADT7 for Y2H assays. Three pGBK fusion constructs (MADS68 1-59 , MADS68 1-87 , and MADS68  ) and the pGADT7 fusion construct were co-transformed into Y2HGOLD and plated on the SD-Trp-Leu medium. PCR was used to verify the viability of the positive colonies, which were then subjected to further testing on the SD-Trp-Leu and SD-Leu-Trp media. After three or four days, images are captured.

Firefly luciferase complementation assays
The N. benthamiana leaves were tested using the firef ly luciferase complementation assay at the six-week mark, following the protocol established earlier [75]. Full-length CDSs of AcMADS68, AcMYBF110, AcMYB123, and AcbHLH1 were inserted into the binary vector pCambia1300cLUC (35S:CLuc) or pCambia1300nLUC (35S:NLuc). Preparation, as well as infiltration, of Agrobacterium were performed following the protocol for the transient expression assay. We used the Steady-Glo Luciferase Assay System (Promega) to measure the firef ly luciferase activity of the samples.

Co-Immunoprecipitation (Co-IP) assay
CDs of AcMYBF110 and AcMYB123 were cloned into the pCAMBIA35s-4 × Myc-MCS-3 × FLAG vector. AcMADS68-GFP was also used in the Co-IP assay. The leaves of N. benthamiana were harvested 48 hours after being co-infiltrated with various Agrobacterium mixtures. Anti-Flag antibodies (TransGen, Haidian District, Beijing, China) and anti-GFP antibodies (TransGen, Haidian District, Beijing, China) were used in Co-IP experiments.

Yeast one-hybrid (Y1H)
We cloned the promoter of AcbHLH1 into pAbAi-baits (Clontech) and the CDS of AcMADS68 into the pGADT7 vector. After transforming the linearized pAbAi-baits into Y1H Gold, the bait strain's minimal inhibitory concentration for Aureobasidin A (AbA) was determined by using the SD/-Ura and SD/-Ura/AbAx medium. Next, yeast cells harboring bait constructs were transfected with either the empty vector (AD) (the control) or AD-AcMADS68 and thereafter spotted on SD/−Leu medium with the least amounts of the AbA antibiotic after being diluted with a 10-fold dilution cycle.